Mitochondria are essential organelles found in most eukaryotic cells and provide for diverse metabolic functions. Fully active mitochondria require proteins and RNAs encoded both by the mitochondrial and nuclear genomes. In humans, at least 600 nuclear encoded proteins are imported into mitochondria and another 13 proteins are encoded by mitochondrial genes. The translation of this limited number of mitochondria-encoded proteins requires mitochondrial ribosomes, translation factors and transfer RNAs. Recent studies, of evolutionary diverse organisms, have shown that some or all of the mitochondrial tRNAs can be nuclear-encoded and must be imported into the mitochondria. In trypanosomes, all mitochondrial tRNAs are nuclear encoded and most are localized to both the mitochondrion and cytosol. While remarkable progress has been made in the elucidation of the mechanism of protein import into fungal and mammalian mitochondria, little is known about the mechanism of mitochondrial RNA import. The overall goal of this proposal is to dissect the pathway of tRNA biosynthesis and trafficking in African trypanosomes. To accomplish this goal the following specific aims are proposed. In Specific Aim I, the biosynthetic pathway for trypanosome tRNAs will be investigated by in vitro transcription and kinetic analysis of tRNA trafficking in intact cells. In Specific Aim II, the role of specific sequence and/or structural motifs in tRNA export from the nucleus and import into the mitochondria will be determined. Finally, in Specific Aim III, trypanosome proteins that interact with precursor and mature tRNAs to influence tRNA trafficking and ultimately mitochondrial import will be identified. Trypanosomes offer an attractive model to study tRNA trafficking pathways since both in vitro and in vivo systems have recently been developed to study both tRNA synthesis and mitochondrial import. In addition, mitochondrial tRNA import may have therapeutic potential since, in contrast to trypanosomes, all mammalian mitochondrial tRNAs are encoded by mitochondrial genes.